Methods for purifying nucleic acids

ABSTRACT

Methods are provided for producing highly purified compositions of nucleic acids by using a highly streamlined and readily automated process. The methods use static mixers for lysing cells and precipitating debris, following by centrifugation and ion exchange chromatography. The process may include a purification step using tangential flow ultrafiltration. A scaleable process for producing pharmaceutical grade plasmid DNA, useful for gene therapy, is provided.

CLAIM OF PRIORITY

This United States Utility Patent Application filed under 35 U.S.C. §120and 37 C.F.R. 1.53(b) is a continuation of U.S. patent application Ser.No. 09/121,798, filed Jul. 23, 1998, now U.S. Pat. No. 7,807,822 whichis a continuation-in-part of U.S. patent application Ser. No.08/691,090, filed Aug. 1, 1996, now U.S. Pat. No. 6,011,148, issued Jan.4, 2000. The above application and patent are each herein incorporatedfully by reference.

FIELD OF THE INVENTION

The invention relates to methods for producing high purity nucleicacids. The invention relates in particular to methods for preparingpharmaceutical quality nucleic acids.

BACKGROUND OF THE INVENTION

Since the advent of recombinant DNA, methods have been developed andimproved for the purification of DNA and RNA to further molecularbiology research. While these methods have allowed considerable study ofnucleic acids in research environments, they have not addressed issuesinvolved in the human clinical use of purified nucleic acids such as isrequired for many current gene therapy protocols.

Gene therapy involves the introduction of nucleic acid into a patient'scells, which, when expressed, provide a therapeutic benefit to thepatient. Examples include the introduction of an exogenous, functionalgene to correct a genetic defect in a patient carrying a defective geneor to compensate for a gene that is not expressed at sufficient levels.Other examples include the introduction of mutant genes, antisensesequences or ribozymes to block a genetic function, e.g., in thetreatment of viral infections or cancer.

Much of the focus in gene therapy has been on using viral vectors,especially retroviral vectors, for introducing exogenous nucleic acidinto a patient's cells. To date, most of these protocols have been forex vivo gene therapy, in which the patient's cells are first removedfrom the patient, genetically modified ex vivo, and then returned to thepatient. The alternative to ex viva gene therapy is in vivo genetherapy. In vivo gene therapy refers to the introduction of exogenousgenetic capability directly to the patient where it is taken up by thetarget cells, which then express the introduced gene to produce atherapeutic product. Viral vectors have been used for in vivo genetherapy although their use is associated with a number of drawbacks,e.g. immunogenicity of the viral vector and safety concerns such asinsertional mutagenesis or viral contamination.

Other means of in vivo gene delivery include the introduction of nakedDNA into the target issue of interest, or the use of lipid-mediated DNAdelivery. Typically, introduction of naked DNA will be used when theexogenous genetic capability is to be introduced directly into thetarget issue. By complexing with liposomes or lipids, DNA is compacted,allowing systemic delivery of the lipid/DNA complexes to various tissuesof interest. See PCT patent application WO 93/25673. Lipid/DNA complexescan be targeted to particular tissues by altering the lipid composition,lipid/DNA ratio, mode of delivery, etc.

For any application in which nucleic acid is introduced into a patient,there is a need to produce highly purified, pharmaceutical grade nucleicacid. Such purified nucleic acid must meet drug quality standards ofsafety, potency and efficacy. In addition, it is desirable to have ascaleable process that can be used to produce large quantities of DNA,e.g., in the range of 100s of milligrams to 100s of grams. Thus, it isdesirable to have a process for producing highly pure nucleic acid thatdoes not use toxic chemicals, known mutagens, organic solvents, or otherreagents that would compromise the safety or efficacy of the resultingnucleic acid, or make scale-up difficult or impractical. It is alsodesirable to prepare nucleic acids free from contaminating endotoxins,which if administered to a patient could elicit a toxic response.Removal of contaminating endotoxins is particularly important where thenucleic acid is purified from gram negative bacterial sources, e.g.plasmid or bacteriophage DNA, which have high levels of endotoxins.

The invention described below meets these needs and provides otherrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention is directed to a method for purifying a nucleicacid from cells.

The present invention provides automatable methods for purifying plasmidDNA from cells. The methods involve use of a static mixer to mix thecells with a lysis solution to provide controlled, gentle mixing of thecells with the lysis solution. Static mixers are further used to mix theresulting lysis mixture with a precipitation solution to precipitate outcell debris and other contaminants, including chromosomal DNA. This istypically followed by an additional step of centrifugation to remove theprecipitated material. The methods of the invention are sufficient toprovide a purified DNA solution that does not require complexpurification steps (e.g., ultrafiltration) prior to application to anion exchange chromatography to produce a final product.

When desired, the methods can be readily automated according to wellknown methods by including appropriate computer controls of steps in theprocess to ensure desired results. The invention also providesparticular conditions by which the methods can be used to prepareplasmid DNA in an automated manner.

DEFINITIONS

“Diafiltration” is a mode of operating an ultrafiltration system inwhich the retentate is continuously recycled and diluted with fresh washsolution to replace that removed as permeate. Diafiltration willgenerally provide a cleaner separation of macromolecules retained in theretentate sample while the smaller molecules pass through into thefiltrate. It may also be used to perform solvent removal or bufferexchange in the same step. “Continuous diafiltration” refers to thecontinuous addition of fresh wash buffer as filtration takes place.“Discontinous diafiltration” refers to the repeated steps ofconcentrating the sample by ultrafiltration, and rediluting with buffer.

“Episomal nucleic acids” are extrachromasomal nucleic acids. Suchnucleic acids include DNA, RNA and chimeric DNA/RNA molecules, and maybe from any biological source including eukaryotic and prokaryoticcells, or may be synthetic. Nucleic acids that may be purified includeribosomal RNA, mRNA, snRNAs, tRNA, plasmid DNA, viral RNA and DNA,synthetic oligonucleotides, ribozymes, and the like. Preferred are viralnucleic acids, and plasmid DNAs. Of particular interest are plasmid DNAsencoding therapeutic genes. By “therapeutic genes” is intended toinclude functional genes or gene fragments which can be expressed in asuitable host cell to complement a defective or under-expressed gene inthe host cell, as well as genes or gene fragments that, when expressed,inhibit or suppress the function of a gene in the host cell including,e.g., antisense sequences, ribozymes, transdominant inhibitors, and thelike.

Thus, e.g., viral DNA or RNA may be purified from prokaryotic oreukaryotic viruses, in which the viral particles are initially purifiedfrom cultures or cells permissive for viral infection in accordance withconventional techniques, e.g., from bacterial, insect, yeast, plant ormammalian cell cultures. Extrachromosomal DNAs include autonomouslyreplicating DNAs from a variety of sources including, e.g., mammaliancells (see, e.g., Yates et al., Nature (1985) 313:812-815; Heinzel etal., Mol. Cell. Biol. (1991) 11(4): 2263-2272), plant cells, yeast cells(e.g., 2 μm plasmids), and prokaryotic cells. Plasmid DNA isolated fromprokaryotic cells include naturally occurring plasmids as well asrecombinant plasmids encoding a gene of interest including, e.g., markergenes or therapeutic genes.

A “gel-layer,” refers to a thin gelatinous layer of biomolecules thatcan form on or in an ultrafiltration membrane. The gel layer isgenerally a cohesive, adherent layer of constant solute concentration,also commonly referred to as concentration polarization. It usually willhave some degree of hydraulic permeability depending on the nature ofthe solute forming the layer. “Gel-layer controlled” ultrafiltrationrefers to filtration conditions where the gel layer becomes the limitingfactor to filtrate flow rate, and further pressure increases have littleor no effect. By contrast, “membrane controlled” conditions are those inwhich the filtrate flow rate is controlled by the permeability of themembrane and the applied pressure.

An “open channel” filter is one which does not have a screen in the feedchannel. By contrast, a “screen channel” or “closed channel” is a filterthat has a screen in the feed channel.

“Permeate” refers to that portion of a sample that passes through theultrafiltration membrane, and is also termed the “filtrate.”

“Retentate” refers to that portion of a sample that does not passthrough the ultrafiltration membrane.

“Static mixer” refers to any flow through device which provides enoughcontact time between two or more liquids to allow substantially completemixing of the liquids. Typically, static mixers contain an internalhelical structure which allows the liquids to come in contact in anopposing rotational flow and causes them to mix in a turbulent orlaminar flow. Such mixers are described, for instance, in U.S. Pat. No.3,286,922.

“Tangential flow” or “cross-flow” filtration refers to a filtrationprocess in which the sample solution circulates across the top of themembrane, while applied pressure causes solute and small molecules topass through the membrane.

“Ultrafiltration” refers to a technique to separate particles byfiltration through membranes having pore sizes ranging from about 0.001μm to about 0.05 μm. Ultrafiltration membranes typically have amolecular weight cut-off (MWCO) in the range of 1,000 to 1,000,000daltons. The MWCO typically is defined as the molecular weight of theglobular solute which is 90% retained by that membrane. See FiltronCatalog, 1995/96, p. 5. The actual molecular weight of particles thatpass through or are retained by a membrane will depend on the size aswell as the conformation and charge of a given molecule, the nature ofthe membrane pore or matrix, and the conductivity and pH of thesolution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a system of the present invention.

FIG. 1B is a schematic diagram of a tangential flow ultrafiltrationprocess.

FIG. 2 illustrates a schematic diagram of large-scale plasmid DNApurification as described in Example 1.

FIG. 3 is a schematic representation of the plasmid p4119.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a highly streamlined process for thepurification of plasmid DNA, which process is readily scalable andyields high quality DNA. The process minimizes complex or expensivepurification steps, thus minimizing cost and allowing an increase inthroughput. It is also an advantage of the invention that the process isreadily automated. The process is particularly suitable for providingpharmaceutical grade plasmid DNA at commercial sale.

In the methods of the invention, static mixers are used to mix certainsolutions, particularly where gentle and complete mixing is desired.Such devices are useful, for instance, in isolating plasmid DNA fromlysed bacterial cells, or for precipitating cell debris, proteins, andchromosomal DNA after lysis. During these procedures, the mixing shouldbe complete to maximize recovery and should be relatively quick tomaintain DNA integrity. If mixing is too vigorous, however, genomic DNAcan be sheared and may contaminate the plasmid preparation. Staticmixers are advantageous in these applications because substantiallycomplete mixing can be obtained while minimizing shear of genomic DNA.In addition, lysis is typically carried out in caustic solutions such asalkali, which can affect the quality of the final preparation. Sincestatic mixers allow continuous flow, the time in contact with thesesolutions can be carefully controlled.

FIG. 1A is a schematic diagram of a system of the present invention. Theillustrated system can be conveniently used for isolation of plasmid DNAfrom bacterial cells. The various solutions are flowed through themixers and lines of the system using pumps (e.g., peristaltic pumps) orpressure according to standard techniques. A tank 10 containing cells islinked by line 20 to static mixer 30. The cells (typically E. coil) areusually first pelleted from a culture according to well knowntechniques. After pelleting, the cells to a density of up to 75% solids,they can be frozen for future plasmid preparation or used directly inthe methods of the invention. Typically, the cells are resuspended in asuitable solution (e.g., 25 mM Tris-HCI, pH 8, 10 mM EDTA, 50 mMdextrose) at room temperature with stirring at 150 rpm for 1 h.Typically, the cells are resuspended in about 5 liters per kilogram (kg)of cell paste. Alternatively, static mixers can be used for thispurpose. RNase can also be added at this time (e.g., 24,000 Kunitz unitsRNase/kg cell paste) to decrease the amount of RNA in later steps.

Tank 40 containing lysis solution is connected to line 20 through line50. The lysis solution can be any solution suitable for lysis of thecells being used. Lysing agents include alkali, detergents, and enzymes.Although organic solvents can be used for this purpose, the use of suchsolvents pose safety and regulatory concerns due to the possibility oftrace amounts in the final product. In addition, such solvents are toxicand inflammable, and pose serious risk and disposal problems if used inthe quantities required for large scale purification. A typical lysissolution will be 0.2N NaOH/1% SDS, and approximately 10 liters per kg ofcell paste is used.

The lysis solution contacts the cell solution in line 20 and the lysismixture proceeds to static mixer 30. The intersection between lines 20and 50 can be adjusted so that the cell solution and lysis solutionenter the static mixer 30 essentially simultaneously. The degree ofmixing is controlled by varying the linear velocity or flow rate of thesolution through the mixer, the type of mixer used, the diameter of themixer, and the number of elements in the mixer. For instance, in thepreparation of plasmid DNA from bacterial cells a laminar flow staticmixer from Kenics is conveniently used and is preferred to turbulentflow static mixers from Kenics or other vendors. The linear velocityused depends on the manufacturer and type of mixer; this controls theReynolds number achieved and how gentle the mixing is. Turbulent mixerscan sometimes be used at low velocities, such that the shear rate is lowenough to avoid damage to the plasmid DNA. However, the shear rate ismore sensitive to changes in linear velocity in the turbulent flowmixers, making them a less robust choice for consistent large scaleproduction.

A linear velocity of 0.38 to 2.3 feet per second gives acceptableproduct quality when using a 2″ diameter, 24 element, laminar flowstatic mixer from Kenics with an overall length of about 6 feet(corresponding to a Reynolds number from 50 to 500). This linearvelocity range permits sufficient mixing to thoroughly lyse the cellsand yet not be so high that genomic DNA is sheared to a size that isproblematic in later purification steps. At a 0.7 feet per second linearvelocity the flow rate in a 2″ diameter mixer is typically 22 liters perminute.

The lysis mixture exiting the static mixer 30 then flows through line 60to static mixer 70. A tank 80 containing a precipitating solution isconnected through line 90 to line 60. The precipitating solution is usedto precipitate proteins, chromosomal DNA and cell debris. Typically, thesolution will contain potassium acetate. A suitable precipitatingsolution is 3M potassium acetate, adjusted to pH 5.5, with acetic acid({tilde over ( )}5M acetate final). As with the first static mixer, theintersection between lines 60 and 90 can be adjusted so that the lysismixture and the precipitation solution enter the static mixer 70essentially simultaneously. Similar linear velocities are used to ensuresufficient mixing to thoroughly precipitate the proteins and cellulardebris and yet not be so high that genomic DNA is sheared to a size thatis problematic in later purification steps. Typically, approximately 5liters of precipitating solution is used per kg of cell paste.

After exiting static mixer 70, the precipitating solution flows throughline 100 to centrifuge 110. In some embodiments, a tank 120 containing abuffer solution is connected to line 100 through line 130. The buffersolution is used to raise the pH of the solution to minimize acidcatalyzed de-purination of the DNA and to condition the material forbinding onto the anion exchange column, e.g., a pH is the range of 6 to9, preferably from 7 to 8.5. A useful buffer solution for this purposeis 1 M Tris. More concentrated solutions can be used to adjust the pH,however the use of a more diluted buffer solution has other benefits. Adiluted buffer reduces the viscosity of the solution going into thecentrifuge to yield better clarity. It also decreases the ionic strengthof the solution, such that it can be loaded directly onto the anionexchange column.

The target ionic strength for loading onto an anion exchange columndepends on the anion exchange resin used. The ionic strength will beless than that at which the DNA binds the resin at sufficientcapacity—e.g., greater than 0.5 grams of DNA per liter of resin.

The precipitation mixture flows into centrifuge 110 to remove theprecipitated material. The precipitation mixture may be put directlyinto the centrifuge or may be buffered before centrifugation. Thecentrifugation is typically carried out at less than 15,000 g in a diskstack or decanting centrifuge, such as an Alfa Laval MBUX 510.Alternately, the outflow of the static mixer may be connected directlyinto the centrifuge to minimize the need for an additional tank and tominimize the processing time. Depending on the cells processed, thefermentation conditions, and the centrifuge used, it may be morebeneficial to add the buffering solution at the outflow of thecentrifuge to achieve optimal clarity. Those conditions may bedetermined empirically. While it is usually preferable to neutralizeprior to centrifugation, in some cases neutralization may dissolve someprecipitate, which then will not be removed by centrifugation and,therefore, will remain as a contaminant. In that case, it is preferableto add the neutralization solution after centrifugation.

It is an advantage of the present invention that cell lysis,precipitation and clarification can be performed in a continuous,automated process by use of static mixers and centrifugation, withappropriate adjustment of flow rates. The flow rate must be sufficientto achieve adequate mixing, but should not be in excess of the rateallowing sufficient residence time in the centrifuge to achieve theseparation, or to shear genomic DNA. The flow rate should not be so slowthat residence time in the centrifuge is too long, which can permitextended contact time with solutions, allowing degradation of theproduct by endonucleases or alkali. Appropriate sizing of the staticmixers, pumps and centrifuge, and selection of flow rate will allowcontinuous operation of the process, while maximizing yield and quality.Preferably, the process is automated to ensure reproducibility. Anexample of conditions allowing continuous operation is described inExample 4 below. Appropriate assays for monitoring product quality,which are, therefore, useful in optimizing process conditions, are alsodescribed in the Examples that follow.

Ion exchange chromatography may be used to further purify the nucleicacid, particularly from contaminating endotoxin, trace proteins, andresidual cellular contaminants. A chromatography column is packed withan anion exchange chromatography resin. The optimal capacity of thecolumn will be determined empirically based on the resin used and thesize of nucleic acid to be purified.

Ion exchange chromatography resins are commercially available, includingfrom EM Separations (Gibbstown, N.J.), BioSepra (Marlborough, Mass.),Polymer Laboratories (Amherst, Mass.), Perseptive Biosystems (Cambridge,Mass.), Toso Haas (Montgomeryville, Pa.) and Pharmacia (Uppsala,Sweden). For most plasmid DNAs, preferred resins are those with no poreor with a large pore size, e.g., greater than 1000 Å, preferably around3000 Å to 4000 Å; with a medium bead size, e.g., about 20 to 500 μmdiameter; that do not leach matrix components. Ideally, the resin isalso washable, e.g., with sodium hydroxide to allow repeated use.

A chromatography column is packed with an anion exchange chromatographyresin. The optimal capacity of the column is determined empiricallybased on the resin used and the size of the nucleic acid to be purified.The column is packed under low pressure, typically less than about 0.7bar. The pressure will depend on the resin used, and will usually beaccording to the manufacturer's specifications. Normal column operatingpressure may be lower where the resin pore size is smaller, to limittrapping of the nucleic acid in the resin pores. Thus, for resinswithout pores, column operating pressure may be increased. The column ispacked at about twice the anticipated flow rate in accordance withconventional techniques.

The nucleic acid sample is loaded onto the column in a loading buffercomprising a salt concentration below the concentration at which thenucleic acid would elute from the column. Typically, the saltconcentration will be about 10 to 50 mS/cm, depending on the resin used.For weaker anion-exchange resins, a lower conductivity solution will beused, whereas for stronger anion-exchange resins, a higher conductivitysolution will be used. The column will then be washed with severalcolumn volumes of buffer to remove those substances that bind weakly tothe resin. The nucleic acid is then eluted from the column using eitherone or more step increases in the saline concentration or a shallowcontinuous saline gradient according to conventional methods, e.g.,using up to 1.5M NaCl in a Tris (pH 8.5) buffer. Collection of thenucleic acid from the step elution method is accomplished by directlypooling the plasmid into a vessel, based on absorbance, conductivity,volume or time. For eluant from the continuous gradient method, similarmonitoring can be used or sample fractions can be collected andanalyzed. For intermediate scale preparations (e.g., from about 100 mgto about 3 grams nucleic acid), fractions will typically be at least 50ml to 2 liters where the nucleic acid peak is expected, then increasedin volume past the expected peak. Analytical determinations of nucleicacid yield and purity are performed on each fraction. In addition,Limulus ameobocyte lysate (LAL) analyses may be performed on eachfraction to determine residual endotoxin levels in each fraction.Fractions containing high levels of nucleic acid and low endotoxin arepooled. For large scale preparation, step elution is preferred. Forexample, when using a resin as described in the Examples herein, theplasmid DNA loading solution conductivity is about 50 mS/cm, and theplasmid DNA is eluted at about 59 mS/cm. The resulting nucleic acidsample may again be filtered through 0.2 μm filter to control the numberof microbial organisms.

The preferred method for purifying plasmid DNA at larger scale is toload the lysate directly onto the ion exchange chromatography columnafter clarification and neutralization. This can be done to avoidadditional purification steps, and greatly simplifies the process. Inthis “direct load” process, after the lysate is clarified bycentrifugation, further debris may be removed by, e.g., decantingthrough a depth filter. The pH and conductivity is then adjusted to theappropriate values, which depend on the anion exchange resin used. Apreferred resin is TMAE Fractogel 650M resin (EM Separations Technology,Wakefield, R.I., US Associate of E. Merck, Darmstadt, Germany). TMAEFractogel 650M is a tentacle ion exchanger having trimethylaminoethylfunctional groups (TMAE) covalently attached to hydroxyl groups of asynthetic methacrylate based polymeric resin backbone. With this resin,the pH is adjusted to about 8.5 and the conductivity is adjusted to lessthan about 50 mS/cm. This can be accomplished by adding about 0.6volumes of 1M Tris per volume of lysate or by diluting 0.25-0.3 foldwith water, then adding Tris base powder to a final concentration of0.62M.

In this embodiment, the ion exchange column is packed and operated at 1column bar pressure, at a linear flow velocity of up to 300 cm/hr. Thecolumn is equilibrated in a Tris buffered, saline solution with aconductivity of about 50 mS/cm. After loading the conditioned lysate,the column is washed with sufficient volumes of 50 mS/cm buffer toremove non-binding impurities, e.g. usually greater than three columnbed volumes of buffer, and preferably about five column bed volumes areused or until an A₂₆₀ reading returns to baseline. Weakly boundimpurities (proteins, RNA and host DNA) are eluted from the column witha buffer that is high enough in saline concentration to removeimpurities, yet not elute the plasmid DNA. Usually, at least threecolumn bed volumes of pre-elution buffer are used, with a conductivityof typically 56 mS/cm. The plasmid DNA is then eluted from the column byincreasing the saline concentration further to a conductivity of about59 mS/cm. The plasmid DNA elutes as a single peak and can be pooledbased on absorbance, time or volume of buffer used. The salineconcentration chosen for the pre-elution and elution buffers depends onthe nature of the plasmid, plasmid impurities, and the resin used.Additionally, the saline concentration of the elution buffer isoptimized to elute the plasmid DNA in the smallest volume, yet stillresolve it from other plasmid degradation products.

In some embodiments, ultrafiltration may be used before the step of ionexchange chromatography. Typically, ultrafiltration is carried out asdescribed in WO 98/05673, which corresponds to U.S. Ser. No. 08/691,090,now U.S. Pat. No. 6,011,148, supra.

In brief, FIG. 1B provides a schematic diagram of a tangential flowultrafiltration process. The feed tank 10 comprises the sample solutionto be filtered. The solution enters the filtration unit 50 through thefeed channel or feed line 20. The circulation pump 30, located in thefeed line 20, controls the solution flow. The filtration unit 50comprises the ultrafiltration membrane. Filtration through theultrafiltration membrane separates the sample solution into a permeatesolution and a retentate solution. The permeate solution exits the unitthrough the permeate channel or permeate line 60. Flow through thepermeate channel may be controlled through a permeate valve located inthe permeate channel 60. The retentate solution passes into theretentate channel or rentate line 90, which is circulated back into thefeed tank 10. Pressure across the ultrafiltration membrane(transmembrane pressure or TMP) is measured by pressure detectors in thefeed channel 40 and in the retentate channel 80. TMP is controlled byadjusting the retentate valve 100. When TFU is performed indiafiltration mode, diafiltration buffer 110 is added to the samplesolution in the feed tank 10. When TFU is used to concentrate the samplesolution, however, diafiltration buffer 110 is not added to the feedtank 10. System control can be manual or automated, with pressuretransducers, flow meters, in-line conductivity meters, and otherfeedback loops.

The ultrafiltration membrane will be selected based on the size andconformation of the nucleic acid to be purified, and typically will havea molecular weight cut-off (MWCO) in the range of 1K to 1,000K Daltons.For many supercoiled plasmid DNAs, ultrafiltration membranes having aMWCO around 300 k to 500 k daltons may be used. For some largerplasmids, however, improved speed, purity and quality of the resultantDNA is obtained when larger MWCO membranes are used. Preferably,therefore, plasmid DNA with sizes ranging from about 2 Kb to 15 Kb arepurified using ultrafiltration membranes having a MWCO of 300K daltons;plasmids ranging from about 15 Kb to about 50 Kb may be purified usingmembranes having a MWCO of 500K daltons; and plasmids of about 40 Kb orlarger may be purified using membranes having a MWCO of 1,000K daltons.With some hollow fiber ultrafiltration devices, e.g., those withsymmetric pores, larger nominal pore sizes may be used. For example,plasmid DNA of us to about 5 Kb can be purified using membranes havingup to 500K daltons MWCO in a hollow fiber device.

Under these conditions, plasmid DNA will be retained in the retentatewhile contaminating substances including many proteins, cell membranedebris, carbohydrates, small degraded nucleotides, etc., pass throughthe membrane into the filtrate. Smaller nucleic acids, e.g., smallsynthetic oligonucleotides, may be purified using ultrafiltrationmembranes with a MWCO of around 1K to 5K daltons. For any nucleic acidto be purified, the optimal membrane pore size may be determinedempirically using small scale devices, e.g., centrifugation devices,stirred cell devices, or small scale hollow fiber systems, availablefrom a variety of commercial manufacturers. A manifold system may beused for optimizing parameters in process scale development. Commercialsources for ultrafiltration devices include Pall-Filtron (Northborough,Mass.), Millipore (Bedford, Mass.), and Amicon (Danvers, Mass.).

Many types of ultrafiltration devices useful in the present inventionare commercially available including e.g., a flat plate device, spiralwound cartridge, hollow fiber, tubular or single sheet device. SeeMichaels et al., (1995). Preferably, the ultrafiltration unit is a flatplate device or hollow fiber device.

It has been found that shearing of the nucleic acid is minimalized ifthe filtration device used for TFU is an open-channel device. Screenedchannels inhibit formation of a gel layer and have been found to shearand decrease yield of the retained nucleic acid. Screen channels may bedesigned, however, having minimal compression of the screens such thatthe shearing and nucleic acid loss may be minimized.

The surface area of the ultrafiltration membrane used will depend on theamount of nucleic acid to be purified. Generally, about ten square feetof membrane is used per gram of nucleic acid. Thus, about five squarefeet of membrane is used per 200 to 800 mg nucleic acid; more typically,about five square feet of membrane is used for 400 to about 600 mgnucleic acid.

The membrane may be of a low-binding material to minimize adsorptivelosses, and should be durable, cleanable and chemically compatible withthe buffers to be used. A number of suitable membranes are commerciallyavailable, including e.g., cellulose acetate, polysulfone,polyethersulfone, and polyvinylidene difluoride. Preferably, themembrane material is polyethersulfone.

It has been found that higher yields and purities are obtained when agel-layer is allowed to form at the membrane surface before startingTFU. The amount of time necessary for gel-layer formation may bedetermined empirically by monitoring the permeate solution for productloss, e.g., by HPLC analysis. The gel-layer is adequate once the productloss into the permeate is sufficiently low. Under preferred conditions,the gel layer acts as a second membrane barrier, which may cause nucleicacid molecules which would normally pass through the membrane to beretained. However, it is not necessary to perform the ultrafiltrationunder conditions, e.g., pressure and feed, that are fully gel-layercontrolled. Thus, as used herein, filtration in the presence of a gellayer means that there is sufficient gel layer to cause additionalsolute retention beyond that resulting solely from the ultrafiltrationmembrane.

Typically, the gel layer is allowed to form for approximately 5 to 90minutes, preferably around 20 to 60 minutes, depending on the device andsize of the nucleic acid. For example, using a flat plate device topurify small plasmid DNAs (e.g., 2 Kb), the gel layer will be formed inabout 60 to 90 minutes; for larger plasmid DNAs (e.g., 2-7 Kb), the gellayer will be formed in about 30 minutes. Using a hollow fiber device,the gel layer can be formed in approximately five to 30 minutes for mostplasmid DNAs, or up to 45 minutes for plasmid DNAs less than about 2 Kb.After formation of the gel-layer, the permeate line 60 will be emptiedinto a waste receptacle and filtration allowed to proceed.

If a gel-layer is not allowed to form during an initial circulationperiod, product will usually be lost by leakage into the permeatesolution. The amount of product loss will depend on the type of deviceused, the membrane MWCO and the total amount of nucleic acid in thesample. Thus, in circumstances where product yield is not critical, thefiltration may be performed without an initial circulation period whilethe gel layer forms. In such cases, the gel layer may be allowed to formduring the initial period of filtration, after which product leakageinto the permeate solution will decrease and product will then beretained in the retentate solution. For example, since a gel layer canbe formed in a hollow fiber device in a short time period (e.g., aboutfive minutes for plasmid DNA of about 5 Kb, and about 150 mg DNA/sq.ft.), product loss during the initial period of forming the gel layermay not be significant and, therefore, re-circulation of the permeatesolution into the feed tank while the gel layer forms may not benecessary.

Filtration will be performed using tangential flow to circulate thesample buffer as it crosses the membrane surface. During tangential flowfiltration, pressure is applied across the membrane, which will allowsmaller molecules to pass through the membrane while the retentate isrecirculated. Typically, the flow rate will be adjusted to maintain aconstant transmembrane pressure. Flow rate and pressure will usuallyfluctuate initially due to the formation of a gel layer. Generally,filtration will proceed faster with higher pressures and higher flowrates, but higher flow rate pressures are likely to cause shearing ofthe nucleic acid or loss due to passage through the membrane. Inaddition, various TFU devices may have certain pressure limitations ontheir operation. The pressure, therefore, may be adjusted according tothe manufacturer's specification. For flat plate devices, the pressureis preferably from about 5 psi to about 30 psi, most preferably in therange of 10 psi to 20 psi. For most plasmid DNAs, 15 psi is preferred.Filtration will generally be performed in diafiltration mode.Optionally, the sample solution may initially be filtered without bufferaddition until concentrated to a desired volume. Once concentrated,diafiltration buffer is added and filtration continues to wash theretentate solution of contaminating small molecules and remove unwantedsolvents and salts. Diafiltration may be either continuous ordiscontinuous. Preferably, diafiltration is continuous, and performeduntil from about 510 to about 500 volume equivalents have beenexchanged, preferably about 10 to 100 volume equivalents. Generally,more diafiltration will be performed with increased contaminants boundto the nucleic acids, depending upon the purity required.

Nucleic acids purified by tangential flow ultrafiltration may be useddirectly or may be further purified depending on the level and type ofcontamination in the starting sample and the desired use. Typically, thenucleic acid purified by tangential flow filtration will be greater than90% pure, often 95% to 100% pure as analyzed by HPLC. The nucleic acidthus purified may be used for a number of applications, e.g., molecularbiological applications such as cloning or gene expression, or fordiagnostic applications using, e.g., PCR, RT-PCR, dendromer formation,etc.

For therapeutic uses, e.g. use in gene therapy, it may desirable tofurther purify the nucleic acid obtained from the tangential flowfiltration step. When the tangential filtration step is used, thenucleic acid sample obtained from the tangential flow filtration step issubsequently filtered through a 0.2 μm filter, further purified usingion exchange chromatography, and, optionally, filtered again through a0.2 μm filter. Desirably, the nucleic acid is further concentrated anddiafiltered using tangential flow ultrafiltration, and filtered againthrough 0.2 μm filter as a final sterilization step.

Filtration through 0.2 μm filters, from certain vendors, can be used toremove endotoxin as well as microoganisms, while resulting in minimalnucleic acid loss. 0.2 μm filters are available from a variety ofcommercial sources including, e.g., Pall-Filtron (East Hills, N.Y.),Sartorius (Edgewood, N.Y.), and Gelman (Ann Arbor, Mich.). Ideally, thefilter used will be one that binds endotoxin while allowing nucleic acidto pass through. Pall Ultipor N₆₆ filters and Sartorius Sartorpure GFfilters have been found to remove substantial endotoxin with high yieldof nucleic acid. Preferably, the nucleic acid solution is pre-filteredthrough a nominal 0.2 μm or a 0.45 μm or larger filter before filtrationthrough an absolute 0.2 μm filter. Glass and nylon filters arepreferred. Filters made for the removal of endotoxin, e.g., ion exchangefilters, in many cases are not suitable for use with nucleic acidpurification because the nucleic acid will bind to the filter.

For many applications it will be desirable to further purify the nucleicacid, lower the salt concentration of the resulting nucleic acid sample,concentrate the sample, and/or exchange the buffer to a more suitablebuffer for subsequent uses. A final diafiltration step may be performedat this stage to achieve that result. If desired, a small MWCOultrafiltration membrane may be used for this subsequent diafiltrationstep than used previously for purification, since the nucleic acid willbe highly purified at this stage and predominantly small solutemolecules will be passed through the membrane into the filtrate. Formany plasmid DNAs, a 150K to 100K MWCO membrane may be used with a flatplate device, or a 100K MWCO membrane may be used with a hollow fiberdevice. Hollow fiber devices with about a 100K MWCO membrane arepreferred at this stage, particularly when handling concentrated nucleicacid solutions, due to smaller hold-up volumes, increased flux, higheryields and shorter processing times. Where DNA purified according to theabove protocol is to be complexed with a lipid carrier for use in genetherapy, it is desirable to exchange the DNA into a low conductivitybuffer, preferably by diafiltration. A low-conductivity buffer is meantto include any buffer of less than about 10 mS, preferably less thanabout 1 mS.

At a variety of places in the above protocol, analytical determinationof nucleic acid yield and purity are advantageously performed.Typically, such assays are performed before and after each purificationstep, as well as to each nucleic acid-containing fraction from, e.g.,preparative ion exchange chromatography. Preferred means for performingthese analytical determinations include HPLC analysis of purity,spectrophotometric estimation of yield, silver staining and SDS-PAGE forprotein analysis, and agarose gel electrophoresis and Southern blottingfor DNA analysis.

The following examples illustrate certain aspects of the above-describedmethod and advantageous results. The following examples are shown by wayof illustration and not by way of limitation.

EXAMPLES Example 1 Preparation of p4119 DNA

Pharmaceutical quality DNA was prepared as follows, using asepticculture conditions for all cell culture procedures. FIG. 2 is aschematic representation of the procedural steps.

An inoculum of E. coli containing plasmid p4119 (FIG. 3) was preparedfrom frozen stock by the addition of 1 ml of frozen (−80° C.) bacterialculture to a 500 ml foam-plugged flasks containing 100 ml TB broth(Sambrook et al., 1989) supplemented with carbenicillin (100 μg/ml).Cultures were incubated at 37° C. and shaken at 220 rpm forapproximately 6 hours. Culture growth was determined by visualinspection or by determining OD₆₀₀, whereby OD values between 0.5 and 5were deemed acceptable.

5 ml of this culture was used to inoculate each of 4 bioreactorscontaining 10 L TB media supplemented with carbenicillin (100 μg/ml) andwith 1 ml/10 L Mazu DF204 antifoaming agent. These cultures wereincubated at 3.7° C. and stirred initially at about 300 rpm. Thecultures were aerated and dissolved oxygen was controlled via cascadecontrol loops, agitation, airflow, and oxygen enrichment to an averageof about 40% saturation. Cultures were incubated for about 10 to 16 h.After incubation, cell content of each culture was determined by OD₆₀₀;OD₆₀₀ values ranged from 16 to 18. Cells were harvested bycentrifugation in a refrigerated Carr continuous flow centrifuge.

The cell pellets were spread into thin sheets and frozen at −80° C.until used for further plasmid purification. 3.2 Kg of the cell pelletwas resuspended in 16 L Solution I (25 mM Tris-HCI, pH 8, 10 mM EDTA, 50mM dextrose) at room temperature with stirring at 150 rpm for 1 h. RNasedigestion was achieved by the addition of RNase (305 mg RNase/Kg cellpaste) and incubating the solution on ice for 2 hours. Cells were lysedby the addition of the cells to 32 L Solution II (0.2N NaOH/1% SDS) inan ice bath. The solution is stirred using a Bow-Tie Stirrer (ColeParmer, Vernon Hills, Ill.) for 25 min. This solution was thenneutralized and cell debris and chromosomal DNA were precipitated by theaddition of 16 L ice-cold Solution III (3M potassium, 5M acetate, pH5.5). The solution was mixed with a Bow-Tie Stirrer on ice for 25 min.

The precipitated material was removed from the neutralized cell lysissolution by centrifugation. The solution was aliquoted into 1 Lcentrifuge bottles and centrifuged at 5300 rps for 20 min at 2° C. Thesupernatants were then decanted through two layers Miracloth(CalBiochem, La Jolla, Calif.) arranged at 90° to each other, into acontainer at room temperature. The decanted supernatants were thenfiltered through 1.2 and 0.2 μm filters arranged in series. As analternative to centrifugation at this stage, precipitated material maybe removed by filtration through a diatomaceous earth material such asCelite® HYFLO SUPER CEL® (Celite Corp., Lompoc, Calif.). See U.S. Pat.No. 5,576,196.

Filtered materials were then pumped into an ultrafiltration unit and theDNA solution filtered by tangential flow filtration through aPall-Filtron Omega open channel Centrasette unit using 25 ft² ofpolyethersulfone (PES) membrane having a MWCO of 300K. The solution wasintroduced into the unit under a pressure of 10 psi, with the permeatechannel open, and the solution allowed to circulate through the unit forabout 50 min until a gel layer was formed. The permeate channel was thendirected to a waste receptacle, and the DNA solution was filtered at apressure of 10 psi until the solution was concentrated to a volume ofabout 3.6 L. Diafiltration buffer (Tris-HCI, pH 8.5) was then added andthe solution was continuously diafiltered at a pressure of 10 psi, flowrate of about 1 L/min, until approximately 50 volume exchanges wereperformed.

After diafiltration, the retentate was recirculated through theultrafilter for 10 min with the permeate valve closed. The retentate wasremoved and the membrane washed twice by an additional 1 L diafiltrationbuffer per wash for 10 min each, with the permeate valve closed. Thewash solutions were added to the retentate and analyzed by HPLC andOD_(260/280) analysis.

HPLC analyses were performed on a 4.6 mm×3.5 cm HPLC column packed withTSK-GEL DEAE-NPR resin at a buffer flow rate of 1 ml/min and monitoredat 254 nm. Samples were diluted 1:20 with Buffer A (20 mM Tris-HCI, pH8) and injected onto the column in a volume of 25 μl. Sample was elutedin a gradient of 0% Buffer B (20 mM Tris-HCI, pH 8/2M KCl): 100% BufferA to 60% Buffer B: 40% Buffer A over 9 min. HPLC analysis of thepermeate, and retentate containing plasmid DNA product showed thatplasmid DNA was typically 95% pure, and often 100% pure as determined byHPLC at this stage.

Spectrophotometric analysis was performed at wavelengths of 250, 260,and 280 nm. Typical ratios for purified DNA are OD₂₆₀/OD₂₅₀>1.1, andOD₂₆₀/OD₂₈₀>1.9. A total of 2.307 g of plasmid DNA was isolated andpurified in the above procedure, having OD₂₆₀/OD₂₅₀ of 1.1047 andOD₂₆₀/OD₂₈₀ of 1.9290.

The recovered plasmid DNA was filtered twice through a Gelman GroundWater Capsule 0.45 μm filter, followed by two filtrations through aPall-Filtron Capsule N₆₆ 0.22 μm filter.

Plasmid DNA was further purified by ion exchange chromatography. DNA wasloaded in a total volume of 4740 ml onto a Amicon Vantage A columnpacked with a 2.5 L bed volume of TMAE-650 (M) (trimethylamino ethyl)Fractogel (EM Separations, Gibbstown, N.J.). The column was equilibratedwith Equilibration Buffer (50 mM Trist, pH 8.5) at a LFV (linear flowvelocity) between 80-150 cm/hr at 0.7 bar column pressure. The DNA wasloaded at 225 ml/min flow rate at 0.7 bar column pressure. The columnwas washed with 3 to 5 column volumes of Equilibration Buffer at 32-35mS. DNA was eluted from the column over an elution gradient of from 32mS to 59 mS or from 0.5M NaCI to 1.5M NaCl in 50 mM Tris, pH 8.5, at aflow rate of 225 ml/min and a column pressure of 0.55 bar. Fractionswere collected in volumes of 130 to 1650 ml starting when the A₂₆₀ wasgreater than 0.2 and ending when A₂₆₀ was less than 0.2.

All fractions were analyzed by HPLC and LAL endotoxin assay. The resultsare shown in Table 1. A total of 2044 mg DNA was loaded onto the columnand 1946 mg were recovered in a total volume of 6079 ml, a yield of95.22%. Column fractions 2-10 were pooled (1905 mg DNA and 3.73×10⁵ EULPS in 5941 ml). Fractions to be pooled were chosen to provide themaximum yield of recovered DNA while minimizing the amount ofcontaminating lipopolysaccharide (LPS) in the preparation.

TABLE 1 DNA conc. Volume Yield LPS LPS/DNA Fraction (mg/ml) (ml) (mg)(EU/ml) (EU/mg) 1 0.30042 137.59 41.33 263.7 878 2 1.04234 531.01 553.49353.2 339 3 0.88851 501.18 445.30 103.2 116 4 0.62513 481.00 300.69 53.685.7 5 0.43912 529.82 232.65 38.8 88.4 6 0.26979 585.56 157.98 35.0 1307 0.14750 526.13 77.60 15.3 104 8 0.08712 568.91 49.56 11.8 135 90.05810 603.83 35.08 9.12 157 10 0.03288 1613.78 53.06 6.49 197

The recovered DNA solution was filtered through a Pall Ultipor N₆₆ 0.2μm filter. To reduce the salt content, the DNA solution was subjected toa final defiltration step using a Pall-Filtron Centramate open channel100K MWCO membrane (2.0 sq. ft.). The filtration unit and membrane werefirst equilibrated with 1 L of a solution of 10 mM Tris-HCI, pH 8.0. Thebuffer is circulated across the membrane using a pump and a sterilereservoir bottle. The DNA solution was added with the permeate channelfully open, and the solution circulated for approximately 30 min at 10psi. The DNA solution was then ultrafiltered until concentrated to avolume of approximately 100 ml. The concentrated solution was thendiafiltered using continuous diafiltration against a solution of 10 mMTris-HCI, pH 8, at 10 psi and permeate flow rate of 120 ml/min until theconductivity of the solution was decreased from an initial value of 35mS to less than 1 mS (0.60) (buffer conductivity=0.53 mS). With thepermeate valve closed, the rententate was then recirculated through theultrafilter for 10 min. The retentate was then collected, and themembrane washed with three 100 ml washes of 10 mM Tris-HCI, pH 8.0.

The DNA and endotoxin concentrations of the diafiltered DNA solution andeach of the washes was determined as above. The retentate and first washwere pooled, yielding 1.366 g DNA at a concentration of 5.564 mg/ml, and90.35 EU/ml or 16.24 EU/mg DNA. This DNA solution was filtered through aMillipore Millipak 40 0.22 μm filter, followed by filtration of anadditional 25 ml of final diafiltration buffer, and the two solutionspooled to yield the final product.

Yield of final plasmid DNA product from the final ultrafiltration was80%. The final product was then aliquotted and stored at −20° C. untiluse. The final product was determined to meet the following QualityControl specifications:

Color clear to slightly cloudy Endotoxin <100 EU/ml Purity >95% by HPLCDNA homogeneity >90% ccc (covalently closed circular) RNA <2% byanalytical HPLC ssDNA <1% by analytical HPLC Protein <0.1% by analyticalHPLC, silver stain and SDS-PAGE Genomic DNA <1% by analytical HPLC andSouthern Blot Conductivity <1 mS pH 7-8.5

Sterility was assayed by day 21 triptose broth culture showing nocolonies. Identity was determined by restriction endonuclease digestionand analysis by agarose gel electrophoresis.

Example 2

The following example shows that either going at too slow a linearvelocity through the static mixer or using too few mixing elements willresult in poor mixing and poor lysis of the cells, leading to lowproduct yields (Table 2 below).

TABLE 2 Effects of different flow velocities (shear rate) as a functionof number of elements. Kenic Shear Pipe I.D. pipe # of VelocityViscosity Rate Mixer Diam (in) Elements (ft/sec) (cps) (sec⁻¹) AfterLysis Pipe 1 0.375 24 0.77 25 590 well mixed 0.375 24 1.50 25 1156 wellmixed 0.375 18 0.77 25 590 some heterogeneity 0.375 18 1.50 25 1156 someheterogeneity After Precipitation Pipe 2 0.620 24 0.37 3 170 uniformed,buoyant ppt 0.620 24 0.74 3 343 uniformed, buoyant ppt 0.620 18 0.37 3170 uniformed, buoyant ppt 0.620 18 0.74 3 343 uniformed, buoyant pptUsing 24 elements is optimal to achieve a well mixed homogenous systemafter the first mixing step (cell lysis by sodium hydroxide/detergent).

Example 3

The following example shows that using too high a linear velocity or toomany mixer elements, especially in the second static mixer, will resultin a finely divided precipitate due to too high a shear rate. The effectof too high a shear rate is that the quantity of contaminating genomicDNA in the final product is increased, and it can cause shear damage tothe plasmid DNA. Table 3 shows the level of genomic DNA contamination,based on analyzing the outflow of the second static mixer by Southernblot analysis.

TABLE 3 Plasmid quality vs. linear velocity. Genomic DNA Actual dataconcentration w Kenic I.D. pipe Shear based pipe Flowrate Diam ViscosityVelocity rate on Southern mixer (lpm) (in) (cps) Reynolds (ft/sec)(1/sec) blot (μg/ml) No >30 mixer (control) w/ >30 impeller (control)Pipe 2 0.66 0.620 3 326 0.19 86  15-30 1.31 0.620 3 646 0.37 171 7.5-152.63 0.620 3 1297 0.74 343 7.5-15 3.94 0.620 3 1944 1.11 514 7.5

Table 3 shows the genomic DNA content as a function of linear flowvelocity in a 24 element static mixer. It appears that as the linearflow velocity increases, genomic content decreases due to increasemixing of the static mixers. Beyond an optimal linear flow velocity ofabout 0.7 to 1.1 ft/sec, however genomic content increases due to highshear rate. Precipitate generation at flow velocities higher thanoptimal show a smaller buoyant precipitate from sheared genomic DNA.

Example 4

The following example shows the effect of mixing velocity on plasmid DNAquality (determined by Southern blot analysis of the outflow from thesecond static mixer). The optimal number of elements depends, in part,on the flow rate. When too few elements are used and the velocity is tooslow, plasmid DNA yield decreases and genomic DNA contaminationincreases. When too few elements are used and the velocity is too high,plasmid DNA yield decreases and plasmid-derived impurities increase.Similar results occur at a desired flow rate if too many elements areused (i.e., too much mixing).

The flow rate is selected to match that desired for centrifugation,while still maintaining high plasmid DNA yield and minimizing genomicDNA contamination. This allows the lysis, precipitation andclarification steps to be performed simultaneously, withoutinterruption, and to be automated. In this case, when using laminar flowstatic mixers of the size used in this Example, the optimal flow rate isin the range of 0.7 to 1.1 ft/sec, with 24 elements.

TABLE 3 Actual Data w I.D. Kenic Flow pipe Ve- Shear pipe rate diamReyn- locity # of rate Genomic mixer (lpm) (in) olds (ft/sec) Elements(1/sec) (ng/ul) no >30 mixer control Pipe 1 0.25 0.375 24 0.19 12147 >60 1.00 0.375 98 0.77 12 590 >60 3.00 0.375 294 2.30 12 1769 30-45Pipe 2 0.33 0.375 269 0.25 12 195 1.31 0.375 1068 1.01 12 772 3.94 0.3753213 3.03 12 2323 Pipe 1 0.25 0.375 24 0.19 36 147 30-45 Pipe 2 0.330.375 269 0.25 36 195 Pipe 1 3.00 0.375 294 2.30 36 1769 >60 Pipe 2 3.940.375 3213 3.03 36 2323

Example 5 Purification by Direct Load of Clarified Lysate on to AnionExchange Chromatography Column

Approximately 40 L of fermentation broth yields about 2.2 kg of cellpaste. After re-suspension of the cell paste, lysis and precipitation,approximately 40 liters of solution were ready for clarification bycentrifugation. Centrifuging in a non-continuous centrifuge (SorvallRC3b) at 7500×g for 25 minutes removed the solids and yielded aclarified product. Tris base (solid) was added to adjust the pH of theclarified product to 8.5 (a final concentration of 0.67 M). After Trisbase addition, the conductivity decreased from 53 mS/cm to 50 mS/cm. Theneutralized lysate was filtered in series with a nominal 0.2 μm glassfilter (Sartorpure GF) and an absolute 0.2μ nylon filter (Pall UltiporN₆₆) (5 ft² each) to reduce bacterial load and endotoxin levels.

The filtered lysate was then directly loaded (155 cm/hr linear flowvelocity) onto an equilibrated (sodium chloride, 50 mM Tris, pH 8.5, 50mS/cm) 1.5 liter TMAE Fractogel 650M column (13 cm diameter, 13 cmheight) (EM Separations Technology, Wakefield, R.I., US Associate E.Merck, Darmstadt, Germany). The column was then washed withequilibration buffer for five column volumes (the A₂₆₀ reading was backto baseline). The column was further washed with three column volumes of50 mM Tris-HCL, 1M NaCl pH 8.5 (56 mS/cm) to elute the endotoxin andgenomic DNA. The plasmid DNA was then step eluted from the column using50-90 mS/cm conductivity Tris/NaCl. The fractions containing highplasmid DNA levels were pooled.

The pooled product contained 1787 mg DNA, endotoxin level of 16 EU/mg,and 1.6% genomic DNA. The product was filtered again through 0.2μnominal glass and 0.2μ absolute nylon filters described above. Afterfiltering the product contained endotoxin level of 1 EU/mg and 0.18%genomic DNA, with 94% yield. The filtered product was diafiltered andsubjected to a final 0.2μ sterilization filter as described inExample 1. The final product was 5.6 mg/ml, with 0.4 EU/mg and less than0.2% genomic DNA.

The examples and embodiments described herein are for illustrativepurposes only, and various modifications will be apparent to those ofskill in the art, the invention to be limited only by the scope of theappended claims. All publications, patents and patent applications citedherein are hereby incorporated by reference as if set forth in theirentirety herein.

What is claimed is:
 1. A method for purifying plasmid DNA from asolution containing plasmid DNA, comprising the steps: a) contactingsaid solution with a precipitation solution; b) flowing the solutionobtained in step a) through a static mixer under conditions of flow thatprovide Reynolds numbers in the range of about 50 to 500; c) furtherpurifying said plasmid DNA using an open tangential flow ultrafiltration(TFU) under conditions that promote formation of a gel layer and saidplasmid DNA is retained in a retentate solution; d) contacting saidretentate solution obtained in step c) with an ion exchangechromatography resin under conditions in which plasmid DNA is bound tosaid resin; and e) eluting said plasmid DNA from said resin using a stepgradient or a continuous gradient of a salt solution with increasingconductivity.
 2. The method of claim 1, further comprising exposing saidsolution obtained by step c) to a ribonuclease (RNAase).
 3. The methodof claim 1, said ion exchange chromatography resin being an anionexchange chromatography resin.
 4. The method of claim 1, said ionexchange resin being a strong anion exchange chromatography resin. 5.The method of claim 1, wherein said TFU is carried out using anultrafiltration membrane having a molecular weight cutoff (MWCO) of 300K Daltons, said plasmid DNA having sizes in the range of 2 Kb to 15 Kbbeing retained in said retentate solution.
 6. The method of claim 1,wherein said TFU is carried out using an ultrafiltration membrane havinga MWCO of 500 K Daltons, said plasmid DNA having sizes in the range of15 Kb to 50 Kb being retained in said retentate solution.
 7. The methodof claim 1, wherein said TFU is carried out using an ultrafiltrationmembrane having a MWCO of 1000 K Daltons, said plasmid DNA having a sizegreater than 40 Kb being retained in said retentate solution.